Dr Anestis Tsakiridis

Welcome to the laboratory of Dr Anestis Tsakiridis.

Our group aims to define the molecular basis of cell fate decisions during embryonic development through the use of human pluripotent stem cells (hPSCs).

We focus particularly on the in vitro modelling of the events guiding the “birth” of the spinal cord and trunk musculature from a common precursor population known as Neuromesodermal Progenitors (NMPs).

Our ultimate goal is to achieve the precise and efficient engineering of NMP-derived cell types for regenerative medicine and disease-modelling applications.

Neurons


Research

Various studies in different vertebrate organisms have revealed the existence of a bipotent stem cell-like population which drives embryonic axis elongation through the coordinated production of posterior neuroectoderm (PNE) and paraxial mesoderm (PXM), the building blocks of the spinal cord and trunk skeletal muscle/skeleton respectively. In the mouse embryo these Neuromesodermal Progenitors (NMPs) reside in the late primitive streak (PS) area and later the tailbud, sites which exhibit high WNT/FGF signalling activity.

Research image 1

NMPs are marked by the co-expression of the transcription factors (TFs) Brachyury (T(BRA)) and SOX2, which are indicative of a mesodermal and neural character respectively. Apart from being an excellent model for studying the mechanisms underlying cell fate (neuroectoderm vs mesoderm) decision-making, NMPs also comprise an attractive source for generating spinal cord cells and skeletal muscle in vitro. These cell types are currently difficult to derive from pluripotent stem cells (PSCs) probably because conventional directed differentiation protocols are heavily influenced by the idea that mesodermal and neuroectodermal lineages arise from separate precursors and ignore the existence of NMPs. Thus the ability to maintain pure cultures of NMPs in vitro and define the optimal conditions for their differentiation would be highly desirable.

However, the limited availability of micro-dissected embryonic NMPs has been a major obstacle to the study of both their biology and the molecular events driving their differentiation. To address this issue we have recently defined the optimal culture conditions for the induction of functional adherent NMPs from mouse and, importantly, human PSCs (hPSCs) involving treatment with the WNT agonist CHIR99021 (CHIR) and FGF2. Under these conditions, the majority of the resulting cultures consist of T(BRA)+SOX2+ NMPs after 48-72 hours of treatment.

We now aim to use hPSC-derived NMPs as a tractable in vitro system to address the following questions:

  1. How do embryonic cells proceed from pluripotency to bipotency?
  2. What are the molecular determinants of a NM bipotent state?
  3. How do NMPs generate PNE and PXM cells
  4. What are the optimal culture conditions for the efficient production of posterior (thoracic and lumbosacral) spinal cord cell types from NMPs?
  5. Are “altered” NM bipotent states linked to disease?

Our research employs hPSC culture and differentiation, CRISPR/Cas9-based genome editing, next generation sequencing and high content/live imaging.

Funding: The University of Sheffield,  Biotechnology and Biological Sciences Research Council (BBSRC), The Royal Society, Children's Cancer and Leukaemia Group/Little Princess Trust

Publications

  1. Economou, C*., Tsakiridis, A*, Wymeersch, F., Gordon-Keylock, S., Dewhurst, R. E., Medvinsky, A., Smith, A. J. H. & Wilson, V. (2015).
    Intrinsic factors and the embryonic environment influence the formation of extragonadal teratomas during gestation.
    BMC Dev Biol 15:35
    *joint first authors
  2. Tsakiridis, A* & Wilson, V*. (2015).
    Assessing the bipotency of in vitro-derived neuromesodermal progenitors
    [v2; ref status: indexed, http://f1000r.es/5p3] F1000Research 2015, 4:100 (doi: 10.12688/f1000research.6345.2).
    *joint corresponding authors
  3. Gouti, M*., Tsakiridis, A*., Wymeersch, F., Huang, Y., Kleinjung, J., Wilson, V. & Briscoe, J. (2014).
    In vitro generation of neuromesodermal progenitors reveals distinct roles for Wnt signalling in the specification of spinal cord and paraxial mesoderm identity.
    PloS Biology. Aug 26;12(8):e1001937. doi: 10.1371/journal.pbio.1001937. eCollection 2014 Aug.
    *joint first authors
  4. Tsakiridis, A*., Huang, Y., Blin, G., Skylaki, S., Wymeersch, F., Osorno, R., Economou, C., Karagianni, E., Zhao, S., Lowell, S. & Wilson, V*. (2014).
    Distinct Wnt-driven primitive streak-like populations reflect in vivo lineage precursors.
    Development 141, 1209-1221 (One of the three most-read Development articles in 2014).
    *joint corresponding authors
  5. Huang, Y., Osorno, R., Tsakiridis, A. & Wilson, V. (2012).
    The in vivo differentiation potential of epiblast stem cells revealed by chimeric embryo formation.
    Cell Reports 2, 1571-1578 (f1000 recommended).
  6. Osorno, R*., Tsakiridis, A*., Wong, F., Cambray, N., Economou, C., Wilkie, R., Blin, G., Scotting, P. J., Chambers, I. & Wilson, V. (2012).
    The developmental dismantling of pluripotency is reversed by ectopic Oct4 expression.
    Development 139, 2288-2298 (f1000 recommended).
    *joint first authors
  7. Brickman, J.M., Tsakiridis, A., To, C. & Stanford, W.L. (2010).
    A wider context for gene trap mutagenesis.
    Methods Enzymol. 477, 271-95.
  8. Tsakiridis, A., Tzouanakou, E., Rahman, A., Colby, D., Axton, R., Chambers, I., Wilson, V., Forrester, L. & Brickman, J.M. (2009).
    Expression-independent gene trap vectors for random and targeted mutagenesis in embryonic stem cells.
    Nucleic Acids Res. 37, e129.
  9. Tsakiridis, A., Alexander, L.M., Gennet, N., Livigni, A., Sanchez-Martin, R.M., Li, M., Bradley, M. & Brickman, J.M. (2009).
    Microsphere-based tracing and molecular delivery in embryonic stem cells.
    Biomaterials 30, 5853-61.
  10. Sanchez-Martin, R.M., Alexander, L.M., Muzerelle, M., Cardenas-Maestre, J.M., Tsakiridis, A., Brickman J.M. & Bradley, M. (2009).
    Microsphere-mediated protein delivery into cells.
    Chembiochem. 10, 1453-6.
  11. Tsakiridis, A., Tzouanacou, E., Larralde, O., Watts, T.M., Wilson, V., Forrester, L. & Brickman, J.M. (2007).
    A novel triple fusion reporter system for use in gene trap mutagenesis.
    Genesis 45, 353-360.
Positions

There are currently no positions available in the lab. However, we always welcome speculative applications. Please email your CV and research interests to a.tsakiridis@sheffield.ac.uk

Members

Anestis Tsakiridis (Group leader)

Anestis TsakiridisAnestis Tsakiridis did his PhD in Lesley Forrester’s laboratory (2002-2006) at the University of Edinburgh focusing on the development of novel gene trap vectors for random inactivation of lineage-specific genes in mouse embryonic stem cells (mESCs).

He then joined Josh Brickman’s group at the Institute for Stem Cell Research in Edinburgh where, in collaboration with Mark Bradley’s Combinatorial Chemistry group, worked on the development of a novel microsphere-based approach for high-throughput delivery of macromolecules into ESCs. In 2009 he switched his focus on the study of early embryonic development by joining the group of Val Wilson (MRC Centre for Regenerative Medicine, Edinburgh).

As part of the Wilson group he defined decreasing Oct4 expression as the major factor contributing to the developmental extinction of post-implantation embryonic pluripotency and demonstrated that ectopic Oct4 reactivation is sufficient to re-instate pluripotency in non-pluripotent somatic cells. He also explored how Wnt signalling acts on pluripotent cells to drive the progressive generation of bipotent primitive streak lineage precursors using mouse epiblast stem cells (mEpiSCs) as an in vitro model of the gastrula stage embryo and showed for the first time that neuromesodermal progenitors (NMPs) can be “captured” in vitro. He expanded this finding by optimizing the culture conditions for the efficient derivation of NMPs from mEpiSCs and, for the first time, human ESCs.

In the summer of 2015 he was awarded a Vice-Chancellor’s Fellowship from the University of Sheffield to start his own research group at the Centre for Stem Cell Biology (CSBC), Biomedical Sciences Department (BMS) in January 2016.

Joseph Kelleher (postdoc)

Erin Stout (MSc student)

Emma Brudenell (Msc student)